Transposon nucleic acids comprising a calibration sequence for DNA sequencing

ABSTRACT

Transposon nucleic acids comprising a transposon end sequence and a calibration sequence for DNA sequencing in the transposon end sequence. In one embodiment, the transposon end sequence is a Mu transposon end. A method for the generation of DNA fragmentation library based on a transposition reaction in the presence of a transposon end with the calibration sequence providing facilitated downstream handling of the produced DNA fragments, e.g., in the generation of sequencing templates.

This application claims priority to U.S. application Ser. No. 61/509,691filed Jul. 20, 2011, which is expressly incorporated by reference hereinin its entirety.

The invention relates to the field of high throughput multiplex DNAsequencing. The invention is directed to transposon nucleic acidscomprising a transposon end sequence and a calibration sequence for DNAsequencing in the transposon end sequence. In one embodiment, thistransposon end sequence is a Mu transposon end. The invention is alsodirected to a method for generation of a DNA fragmentation library basedon a transposition reaction in the presence of a transposon end with thecalibration sequence, providing facilitated downstream handling of theproduced DNA fragments, e.g., in the generation of sequencing templates.

BACKGROUND

“DNA sequencing” generally refers to methodologies aiming to determinethe primary sequence information in a given nucleic acid molecule.Traditionally, Maxam-Gilbert and Sanger sequencing methodologies havebeen applied successfully for several decades, as well as apyrosequencing method. However, these methodologies have been difficultto multiplex, as they require a wealth of labor and equipment time, andthe cost of sequencing is excessive for entire genomes. Thesemethodologies required each nucleic acid target molecule to beindividually processed, the steps including, e.g., subcloning andtransformation into E. coli bacteria, extraction, purification,amplification, and sequencing reaction preparation and analysis.

So called “next-generation” technologies or “massive parallelsequencing” platforms allow millions of nucleic acid molecules to besequenced simultaneously. The methods rely on sequencing-by-synthesisapproach, while certain other platforms are based onsequencing-by-ligation technology. Although very efficient, all of thesenew technologies rely on multiplication of the sequencing templates.Thus, for each application, a pool of sequencing templates needs to beproduced. A major advancement for template generation was the use of invitro transposition technology. The earliest in vitrotransposition-assisted sequencing template generation methodology(Tenkanen U.S. Pat. No. 6,593,113) discloses a method in which thetransposition reaction results in fragmentation of the target DNA, andthe subsequent amplification reaction is carried out in the presence ofa fixed primer complementary to the known sequence of the target DNA anda selective primer having a complementary sequence to the end of atransposon DNA.

In vitro transposition methodology has also been applied to “nextgeneration” sequencing platforms. Grunenwald (U.S. Patent Application20100120098) disclose methods using a transposase and a transposon endfor generating extensive fragmentation and 5′-tagging of double-strandedtarget DNA in vitro. The method is based on the use of a DNA polymerasefor generating 5′- and 3′-tagged single-stranded DNA fragments afterfragmentation without performing a PCR amplification reaction.

Many “next-generation” sequencing instruments require a specificcalibration sequence to be read first as a part of the sequence to beanalyzed (e.g. ion torrent PGM and Roche 454 Genome Sequencer FLXSystem). This calibration sequence has known bases in particular orderand it calibrates the instrument so that it is capable ofdifferentiating the signal generated from different bases during the DNAsequencing reaction. It is necessary that each of the sequencingtemplates comprises this calibration sequence.

Methods that facilitate the downstream handling of the fragmented DNAobtained from the transposition step are needed.

SUMMARY

The invention is related to the modification of a transposon endsequence so that it includes a calibration sequence for DNA sequencing.When the transposon end sequence is inserted into the target DNA in thefragmentation reaction, the calibration sequence is simultaneouslyincorporated into the target sequence.

A modified transposon nucleic acid comprising a transposon end sequenceand an engineered calibration sequence for DNA sequencing in thetransposon end sequence, and a kit for DNA sequencing containing themodified transposon nucleic acid.

An in vitro method for generating a DNA library by incubating atransposon complex comprising a transposon nucleic acid and atransposase with a target DNA of interest under conditions for carryingout a transposition reaction, where the transposon nucleic acidcomprises a transposon end sequence recognizable by the transposase,where the transposon end sequence comprises a calibration sequence forDNA sequencing, and where the transposition reaction results infragmentation of the target DNA and incorporation of the transposon endinto the 5′ end of the fragmented target DNA.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. A Petition under 37 C.F.R. §1.84 requesting acceptance of thecolor drawing is being filed separately. Copies of this patent or patentapplication publication with color drawing(s) will be provided by theOffice upon request and payment of the necessary fee.

FIG. 1 is a gel showing a complex formation with MuA transposase andvarying transposon end sequences.

FIGS. 2A-C show human genomic DNA fragmented with different amounts oftranspososome complexes.

FIG. 3 is a gel showing a complex formation similar to FIG. 1, but withtransposon end of SEQ ID NO: 5.

FIGS. 4A-B are similar to FIGS. 2A-C, but with transposon end of SEQ IDNO: 5.

FIGS. 5A-B are similar to FIGS. 2A-C, but with transposon end of SEQ IDNO: 6.

FIG. 6 shows a transposition reaction with Mu transposon end sequenceson target DNA.

FIG. 7 shows downstream sequencing reaction with target DNA comprisingan incorporated transposon end with calibration sequence.

DETAILED DESCRIPTION

The terms “calibration sequence” or “key sequence” as used hereingenerally refer to a nucleic acid sequence that can be used to calibratea DNA sequencing system. Thus, in embodiments, the particular bases, theorder of the bases, and the number of bases that are present in acalibration sequence depends on the requirements of a particular DNAsequencing system.

In one embodiment, the calibration sequence is a four-nucleotide-longnucleic acid sequence of the four known bases (A, T, C and G) inparticular order incorporated into target DNA to be sequenced. Thecalibration sequence calibrates the sequencing instrument for eachsample so that it is capable of differentiating the signal generatedfrom different bases during the DNA sequencing reaction. For example,sequences TCAG, GTCA and TGCA can be used as calibration sequences.However, the four bases could be presented in the calibration sequencein any possible order. In embodiments, the calibration sequence may belonger than four nucleotides, e.g., the calibration sequence may befive, six, seven, eight, nine, ten, or more nucleotides long. Thecalibration sequence may also comprise bases in addition to, or in placeof, the four known bases A, T, G, and C. For example, the calibrationsequence may contain derivatized and/or artificial nucleotide bases;such modified bases are known to one skilled in the art.

The term “transposon”, as used herein, refers to a nucleic acid segmentthat is recognized by a transposase or an integrase enzyme and is anessential component of a functional nucleic acid-protein complex (i.e.,a transpososome) capable of transposition. In one embodiment, a minimalnucleic acid-protein complex capable of transposition in a Mutransposition system comprises four MuA transposase protein moleculesand a pair of Mu transposon end sequences that are able to interact withMuA (FIG. 6) where the DNA sequences of the fragments from thetransposition reaction are, e.g.,

SEQ ID NO: 1 ............Insert from Target DNA......gap SEQ ID NO: 7SEQ ID NO: 8 gap.....................................................SEQ ID NO: 9and showing the product after gap-filling by a DNA polymerase.

The term “transposase” as used herein refers to an enzyme that is acomponent of a functional nucleic acid-protein complex capable oftransposition and which is mediating transposition. The term“transposase” also refers to integrases from retrotransposons or ofretroviral origin.

A “transposition reaction” as used herein refers to a reaction where atransposon inserts into a target nucleic acid. Primary components in atransposition reaction are a transposon and a transposase or anintegrase enzyme. The method and materials of the invention areexemplified by employing in vitro Mu transposition (Haapa et al. 1999and Savilahti et al. 1995). Other transposition systems can be used,e.g., Tyl (Devine and Boeke, 1994, and WO 95/23875), Tn7 (Craig, 1996),Tn 10 and IS 10 (Kleckner et al. 1996), Mariner transposase (Lampe etal., 1996), Tcl (Vos et al., 1996, 10(6), 755-61), Tn5 (Park et al.,1992), P element (Kaufman and Rio, 1992) and Tn3 (Ichikawa and Ohtsubo,1990), bacterial insertion sequences (Ohtsubo and Sekine, 1996),retroviruses (Varmus and Brown 1989), and retrotransposon of yeast(Boeke, 1989).

A “transposon end sequence” as used herein refers to the nucleotidesequences at the distal ends of a transposon. The transposon endsequences are responsible for identifying the transposon fortransposition; they are the DNA sequences the transpose enzyme requiresto form a transpososome complex and to perform a transposition reaction.For MuA transposase, this sequence is 50 by long (SEQ ID NO. 1)described by Goldhaber-Gordon et al., J Biol Chem. 277 (2002) 7703-7712,which is hereby incorporated by reference in its entirety. Atransposable DNA of the present invention may comprise only onetransposon end sequence. The transposon end sequence in the transposableDNA sequence is thus not linked to another transposon end sequence by anucleotide sequence, i.e., the transposable DNA contains only onetransposase binding sequence. Thus, the transposable DNA comprises a“transposon end” (e.g., Savilahti et al., 1995).

A “transposase binding sequence” or “transposase binding site” as usedherein refers to the nucleotide sequences that are always within thetransposon end sequence where a transposase specifically binds whenmediating transposition. The transposase binding sequence may comprisemore than one site for binding transposase subunits.

A “transposon joining strand” or “joining end” as used herein means theend of that strand of the double-stranded transposon DNA that is joinedby the transposase to the target DNA at the insertion site.

The term “adaptor” or “adaptor tail” as used herein refers to anon-target nucleic acid component, generally DNA, that provides a meansof addressing a nucleic acid fragment to which it is joined. Forexample, in embodiments, an adaptor comprises a nucleotide sequence thatpermits identification, recognition, and/or molecular or biochemicalmanipulation of the DNA to which the adaptor is attached (e.g., byproviding a site for annealing an oligonucleotide, such as a primer forextension by a DNA polymerase, or an oligonucleotide for capture or fora ligation reaction).

Many “next-generation” sequencing instruments (e.g. ion torrent PGM andRoche 454 Genome Sequencer FLX System) require a specific calibrationsequence to be read first as a part of the sequence to be analyzed.Because the in vitro transposition technology is already used tofragment target DNA for sequencing, the disclosed method providestransposon end sequences that include the calibration sequence. In thisway, the calibration sequence would be incorporated to the target DNAduring the fragmentation step. To reduce unusable sequence reads, thiscalibration sequence may be designed as close to the 3′ inserted end ofthe transposon end (i.e., the joining end) as possible.

The MuA transposase recognizes a certain transposon end sequence of 50by (SEQ ID NO:1) but tolerates some variation at certain positions(Goldhaber-Gordon et al., J Biol Chem. 277 (2002) 7703-7712). Variousoptions for including a calibrator sequence into the transposon end weredesigned.

In one embodiment, a modified transposon nucleic acid comprising atransposon end sequence and an engineered calibration sequence for DNAsequencing in the transposon end sequence is provided. The transposonend sequence may be a Mu transposon end sequence, and the Mu transposonend sequence may be any of SEQ ID NOS: 3-6. In one embodiment, the Mutransposon end sequence is SEQ ID NO: 5.

In one embodiment, a modified transposon nucleic acid comprising atransposon end sequence and an engineered calibration sequence for DNAsequencing in the transposon end sequence is provided, where thetransposon end sequence further contains an engineered cleavage site. Anengineered cleavage site in the transposon end sequence can be usefulfor removing parts of the transposon end sequence from the fragmentedDNA, which improves downstream amplification (e.g., by reducingintramolecular loop structures, as a result of less complementarysequence) or reduces the amount of transposon end sequence that would beread during sequencing (e.g., single molecule sequencing). Inembodiments, the engineered cleavage site may be the incorporation of auracil base or a restriction site. Modified transposon end sequencescomprising an engineered calibration sequence for DNA sequencing andoptionally an uracil base or an additional restriction site can beproduced, e.g., by regular oligonucleotide synthesis.

In one embodiment, an in vitro method for generating a DNA library isprovided. The method incubates a transposon complex comprising atransposon nucleic acid and a transposase with a target DNA of interestunder conditions for carrying out a transposition reaction. Transposonnucleic acid comprises a transposon end sequence that is recognizable bythe transposase, and where the transposon end sequence comprises acalibration sequence for DNA sequencing. The transposition reactionresults in fragmentation of the target DNA, and incorporates thetransposon end into the 5′ end of the fragmented target DNA.

In one embodiment, the method further comprises the step of amplifyingthe fragmented target DNA in an amplification reaction using a first andsecond oligonucleotide primer complementary to the transposon end in the5′ ends of the fragmented target DNA. The first and second primeroptionally comprise 5′ adaptor tails.

In one embodiment, the method further comprises the step of contactingthe fragments of target DNA comprising the transposon end at the 5′ endsof the fragmented target DNA with DNA polymerase having 5′-3′exonuclease or strand displacement activity, so that fullydouble-stranded DNA molecules are produced from the fragments of targetDNA. This step is used to fill the gaps generated in the transpositionproducts in the transposition reaction. The length of the gap ischaracteristic to a certain transposition enzyme, e.g., for MuA the gaplength is 5 nucleotides.

To prepare the transposition products for downstream steps, such aspolymerase chain reaction (PCR), the method may comprise the furtherstep of denaturating the fully double-stranded DNA molecules to producesingle stranded DNA for use in the amplification reaction.

In one embodiment, the transposition system used in the method is basedon MuA transposase enzyme. For the method, one can assemble in vitrostable but catalytically inactive Mu transposition complexes inconditions devoid of Mg²⁺ as disclosed in Savilahti et al., 1995 andSavilahti and Mizuuchi, 1996. In principle, any standard physiologicalbuffer not containing Mg²⁺ is suitable for assembly of the inactive Mutransposition complexes. In one embodiment, the in vitro transpososomeassembly reaction may contain 150 mM Tris-HCl pH 6.0, 50% (v/v)glycerol, 0.025% (w/v) Triton X-100, 150 mM NaCl, 0.1 mM EDTA, 55 nMtransposon DNA fragment, and 245 nM MuA. The reaction volume may rangefrom about 20 μl to about 80 μl. The reaction is incubated at about 30°C. for about 0.5 hours to about four hours. In one embodiment, theassembly reaction is incubated for two hours at about 30° C. Mg²⁺ isadded for activation.

In case the transposon end sequence comprises an engineered cleavagesite, the method can comprise a further step of incubating thefragmented target DNA with an enzyme specific to the cleavage site sothat the transposon ends incorporated to the fragmented target DNA arecleaved at the cleavage site. The cleaving enzyme may be anN-glycosylase or a restriction enzyme, such as uracil-N-glycosylase or amethylation specific restriction enzyme, respectively.

In one embodiment, the 5′ adaptor tail of the first and/or second PCRprimer(s) used in the method comprise one or more of the followinggroups: an amplification tag, a sequencing tag, and/or a detection tag.

The amplification tag is a nucleic acid sequence providing specificsequence complementary to an oligonucleotide primer to be used in thesubsequent rounds of amplification. For example, the sequence may beused for facilitating amplification of the nucleic acid obtained.Examples of detection tags are fluorescent and chemiluminescent dyes, agreen fluorescent protein, and enzymes that are detectable in thepresence of a substrate, e.g., an alkaline phosphatase with NBT plusBCIP, or a peroxidase with a suitable substrate. By using differentdetection tags, i.e. barcodes, sequences from multiple samples can besequenced in the same instrument run and identified by the sequence ofthe detection tag. Examples are Illumina's index sequences in TruSeq DNASample Prep Kits, or Molecular barcodes in Life Technologies' SOLiD™ DNABarcoding Kits.

The sequencing tag provides a nucleic acid sequence permitting the useof the amplified DNA fragments obtained from step c) as templates fornext-generation sequencing. For example, the sequencing tag may provideannealing sites for sequencing by hybridization on a solid phase. Suchsequencing tag may be Roche 454A and 454B sequencing tags, AppliedBiosystems' SOLiD™ sequencing tags, ILLUMINA™ SOLEXA™ sequencing tags,the Pacific Biosciences' SMRT™ sequencing tags, Pollonator Polonysequencing tags, and the Complete Genomics sequencing tags.

The detection tag comprises a sequence or a detectable chemical orbiochemical moiety for facilitating detection of the nucleic acidobtained from the amplification step.

In one embodiment, a kit for use in DNA sequencing is provided. The kitcomprises at least a tranposon nucleic acid comprising transposon endsequence and an engineered calibration sequence for DNA sequencing inthe transposon end sequence. In one embodiment, the tranposon nucleicacid is a Mu transposon end sequence. In one embodiment, the Mutransposon end sequence is selected from SEQ ID NOS: 3-6. In oneembodiment, the Mu transposon end sequence is SEQ ID NO:5. In oneembodiment, the tranposon nucleic acid further comprises an engineeredcleavage site. The kit may also comprise additional components, e.g.,buffers for performing transposition reaction, buffers for DNAsequencing, control DNA, transposase enzyme, and DNA polymerase. The kitcan be packaged in a suitable container with instructions for using thekit.

The publications and other materials used herein to illuminate thebackground of the invention, and in particular, to provide additionaldetails with respect to its practice, are incorporated herein byreference. The present invention is further described in the followingexamples, which are not intended to limit the scope of the invention.

EXAMPLE 1 Native Sequence of the Inserted Strand of MuA RecognitionTransposon End

In various embodiments, a calibration sequence is four bases long andcontains each of the four bases (A, T, G, C) sequentially in a row butin any order. The native MuA recognition sequence only contains these atunderlined positions which are far from the 3′-end.

(SEQ ID NO: 1) GTTTTCGCATTTATCGTGAAACGCTTTCGCGTTTTTCGTGCGCCGCTTCAIf these sequences were used as calibrators, a minimum of 34 bases ofthe inserted transposon sequence would have to be read in every targetfragment before reaching the sequence of interest, wasting sequencingreagents and instrument run time. The accuracy of sequence reading alsogradually decreases after each cycle, so the best possible accuracywould always be “wasted” on reading the transposon end sequence. Inaddition, the bases in the “native calibrator sequences” are notnecessarily in the preferred order, as defined by the instruments'requirements. That is why the MuA recognition sequence needs to bemodified near the 3′-end in order to be used in library generation forinstruments that require a calibration sequence. Other transposons, suchas the native Tn5 recognition transposon sequence, do not contain thefour bases in a row anywhere in its sequence.

When the transposon end sequence and MuA transposase were incubatedtogether in a suitable buffer, they formed transpososome complexes (FIG.1). The higher molecular weight DNA band of about 300 by representstransposon end DNA bound to transpososome complexes (mobility in the gelelectrophoresis is retarded due to protein binding) and the smaller DNAband (50 bp) is the free transposon DNA, i.e. not complexed by MuA. M:Molecular weight marker. wt: Native MuA transposon end +MuA transposase.1: Transposon end of SEQ ID NO:4. 2: Transposon end of SEQ ID NO:2. 3:Transposon end of SEQ ID NO:3. −MuA: Control reaction with native MuAtransposon end DNA sequence (SEQ ID NO: 1) without MuA transposase.

When these complexes (formed using wild-type MuA transposon end DNA andMuA transposase) were incubated with target DNA, the transposonsequences were inserted into DNA and the target DNA was fragmented, asshown in FIGS. 2A-C. FIG. 2A: 0.05 g/L MuA in the fragmentationreaction. FIG. 2B: 0.15 g/l MuA. FIG. 2C: 0.2 g/l MuA, gDNA fragmentedwith MuA. Control contains MuA complexes but no gDNA.

TABLE 1 Composition of the complex formation reaction in FIG. 1. Thefollowing components of the complex formation reaction were incubatedfor one hour at 30° C. MuA transposase 0.44 g/l Transposon DNA 3.0 μMTris-HCl pH 8 120.42 mM HEPES pH 7.6 2.6 mM EDTA 1.05 mM DTT 0.10 mMNaCl 102.1 mM KCl 52 mM Triton X-100 0.05 % glycerol 12.08 % DMSO 10 %Different amounts of the complex were incubated with human genomic DNAat 30° C. for one hour (Table 2), and each reaction was replicated eighttimes. The replicates were combined after the fragmentation, DNA waspurified with QIAGEN MinElute PCR Purification Kit, and analyzed withAgilent 2100 Bioanalyzer instrument (FIG. 2).

TABLE 2 Example of final composition of the fragmentation reaction. Forthe fragmentation reaction, the MuA transposon and transposon DNAconcentrations were varied in different experiments, whereas theconcentration of other components was kept constant. MuA transposon 0.05g/l Transposon DNA 0.341 μM gDNA 100 ng Tris-HCl pH 8 40 mM EDTA 0.33 mMNaCl 100 mM MgCl₂ 10 mM Triton X-100 0.05 % glycerol 10 % DMSO 3.3 %

EXAMPLE 2 Changing of the Mu 3′-End to Include the Current Ion TorrentKey Sequence (TCAG)

(SEQ ID NO: 2) GTTTTCGCATTTATCGTGAAACGCTTTCGCGTTTTTCGTGCGCCGCTCAG

This transposon end sequence was tested. MuA transposase did not formcomplexes with this sequence and thus there was no transpositionactivity (see FIG. 1).

EXAMPLE 3 Converting the Fourth T of Mu End Sequence to G (Counting Fromthe 3′-End) to Yield GTCA Key

(SEQ ID NO: 3) GTTTTCGCATTTATCGTGAAACGCTTTCGCGTTTTTCGTGCGCCGCGTCAThis was tested for transposition activity and MuA transposase formedcomplexes with this sequence and was also active (FIG. 1).

EXAMPLE 4 Converting the Third T of Mu End Sequence to G to Yield TGCAKey

(SEQ ID NO: 4) GTTTTCGCATTTATCGTGAAACGCTTTCGCGTTTTTCGTGCGCCGCTGCAThis was tested for transposition activity and MuA transposase formedcomplexes with this sequence and was also active (FIG. 1).

EXAMPLE 5 Modification of the 5th, 6th, and 8th Bases of Mu End Sequenceinto G, A, and T, Respectively, to Yield the Current Ion Torrent Key

(SEQ ID NO: 5) GTTTTCGCATTTATCGTGAAACGCTTTCGCGTTTTTCGTGCGTCAGTTCAThis transposon end sequence worked well with MuA transposase in complexformation, as shown in FIG. 3, which shows complex formation similar toFIG. 1, but with transposon end of SEQ ID NO: 5, and in fragmentation,as shown in FIG. 4, which is similar to FIG. 2, but with transposon endof SEQ ID NO: 5, i.e. showing human genomic DNA fragmented withdifferent amounts of transpososome complexes. FIG. 4A: 0.05 g/L MuA inthe fragmentation reaction. FIG. 4B: 0.15 g/l MuA, with gDNA fragmentedwith MuA and control that contains MuA complexes but no gDNA.

EXAMPLE 6 Modification of the 10th, and 11th Bases of Mu End SequenceInto A and C, Respectively, to Yield

(SEQ ID NO: 6) GTTTTCGCATTTATCGTGAAACGCTTTCGCGTTTTTCGTCAGCCGCTTCA

This transposon end sequence worked well with MuA transposase in complexformation and in fragmentation, as shown in FIG. 5, which is similar toFIG. 2, but with transposon end of SEQ ID NO: 6, i.e. showing humangenomic DNA fragmented with different amounts of transpososomecomplexes. FIG. 5A: 0.05 g/L MuA in the fragmentation reaction. FIG. 5B:0.15 g/l MuA, with gDNA fragmented with MuA and control that containsMuA complexes but no gDNA.

REFERENCES

-   -   Boeke J. D. 1989. Transposable elements in Saccharomyces        cerevisiae in Mobile DNA.    -   Craig N. L. 1996. Transposon Tn7. Curr. Top. Microbiol. Immunol.        204: 27-48.    -   Devine, S. E. and Boeke, J. D., Nucleic Acids Research, 1994,        22(18): 3765-3772.    -   Goldhaber-Gordon et al., J Biol Chem. 277 (2002) 7703-7712    -   Haapa, S. et al., Nucleic Acids Research, vol. 27, No. 13, 1999,        pp. 2777-2784    -   Ichikawa H. and Ohtsubo E., J. Biol. Chem., 1990, 265(31):        18829-32.    -   Kaufman P. and Rio D. C. 1992. Cell, 69(1): 27-39.    -   Kleckner N., Chalmers R. M., Kwon D., Sakai J. and Bolland S.        TnIO and IS10 Transposition and chromosome rearrangements:        mechanism and regulation in vivo and in vitro. Curr. Top.        Microbiol. Immunol., 1996, 204: 49-82.    -   Lampe D. J., Churchill M. E. A. and Robertson H. M., EMBO        J.,1996, 15(19): 5470-5479.

-   Ohtsubo E. & Sekine Y. Bacterial insertion sequences. Curr. Top.    Microbiol. Immunol., 1996, 204:1-26.    -   Park B. T., Jeong M. H. and Kim B. H., Taehan Misaengmul        Hakhoechi, 1992, 27(4): 381-9.    -   Savilahti, H. and K. Mizuuchi. 1996. Mu transpositional        recombination: donor DNA cleavage and strand transfer in trans        by the Mu transposase. Cell 85:271-280.    -   Savilahti, H., P. A. Rice, and K. Mizuuchi. 1995. The phage Mu        transpososome core: DNA requirements for assembly and function.        EMBO J. 14:4893-4903.    -   Varmus H and Brown. P. A. 1989. Retroviruses, in Mobile DNA.        Berg D. E. and Howe M. eds. American Society for Microbiology,        Washington D. C. pp. 53-108.    -   Vos J. C., Baere I. And Plasterk R. H. A., Genes Dev.,        1996,10(6): 755-61.

Applicants incorporate by reference the material contained in theaccompanying computer readable Sequence Listing identified as SequenceListing_ST25.txt, having a file creation date of Jul. 17, 2012 1:49 P.M.and file size of 1.98KB.

What is claimed is:
 1. An in vitro method for fragmenting target nucleicacids comprising contacting the target nucleic acids with a plurality ofMuA transposon complexes, under a condition for transposon-mediatednucleic acid fragmentation, to generate a plurality of nucleic acidfragment products having a calibration sequence added to one end,wherein the MuA transposon complexes contain a first and a secondnucleic acid duplex and a plurality of MuA transposon proteins, whereinthe first and the second nucleic acid duplexes contain (i) a firststrand having the nucleotide sequence of SEQ ID NO:1 that is modified toinclude a calibration sequence near the 3′ end, and (ii) a second strandthat is fully complementary to the first strand, wherein the calibrationsequence is four nucleotides in length and contains any nucleotide basesA, T, C, and/or G in any order and the calibration sequence replaces anyfour nucleotides in the first strand near the 3′ end in a region locatedat position 39-50 of SEQ ID NO:1.
 2. The method of claim 1 wherein thefirst strand comprises the nucleotide sequence of SEQ ID NO:5 whichincludes a calibration sequence 5′-TCAG-3.
 3. The method of claim 1wherein the first strand comprises the nucleotide sequence of SEQ IDNO:3 which includes a calibration sequence 5′-GTCA-3′.
 4. The method ofclaim 1 wherein the first strand comprises the nucleotide sequence ofSEQ ID NO:4 which includes a calibration sequence 5′-TGCA-3′.
 5. Themethod of claim 1 wherein the first strand comprises the nucleotidesequence of SEQ ID NO:6 which includes a calibration sequence5′-TTCA-3′.
 6. The method of claim 1, wherein each of the plurality ofMuA transposon complexes comprises four MuA transposon proteins.
 7. Themethod of claim 1, wherein the calibration sequence added to one end ofthe nucleic acid fragment products is selected from a group consistingof 5′-TCAG-3, 5′-GTCA-3′, 5′-TGCA-3′ or 5′-TTCA-3′.
 8. The method ofclaim 1, further comprising: contacting the plurality of nucleic acidfragment products with a polymerase having 5′ to 3′ exonuclease activityand a plurality of nucleotides.
 9. The method of claim 1, furthercomprising: amplifying the nucleic acid fragment products to producenucleic acid fragment amplicons, by contacting the plurality of nucleicacid fragment products with a plurality of amplification primers, apolymerase, and a plurality of nucleotides.
 10. The method of claim 9,wherein the amplifying includes polymerase chain amplification.
 11. Themethod of claim 9, wherein the plurality of amplification primerscomprise a plurality of tailed amplification primers.
 12. The method ofclaim 9, wherein the plurality of amplification primers comprise adetection tag, selected from a group consisting of a nucleic acidbarcode sequence, a chemical tag, a fluorescent dye, a chemiluminescentdye, or an enzyme.
 13. The method of claim 1, further comprisingsequencing the plurality of nucleic acid fragment products.
 14. Themethod of claim 13, wherein the sequencing comprises a massivelyparallel sequencing method.
 15. The method of claim 9, furthercomprising sequencing the nucleic acid fragment amplicons.
 16. Themethod of claim 15, wherein the sequencing comprises a massivelyparallel sequencing method.